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Abstract

Cardiotoxicity is one of the most significant reasons of attrition in drug development. The present study assessed the sensitivity of various endpoints for early monitoring of drug-induced cardiotoxicity using human embryonic stem cell–derived cardiac cells, including precursors as well as mature cardiomyocytes, by correlating changes in cardiac biomarker expression. Directed differentiation was induced and cardiac progenitor cell (CPC) population were treated with cardiotoxic drugs, namely, doxorubicin (Dox) and paclitaxel (Pac), and with noncardiotoxic drug, namely penicillin G. To assess cardiac-specific toxicity, the changes in the expression of key markers of cardiac lineage, such as Nkx2.5, Tbx5, α-myosin heavy chain α-MHC, and cardiac troponin T, were studied using quantitative real-time polymerase chain reaction (qRT-PCR) and flow cytometry (FC). The half-maximal inhibition in the expression of these cardiac markers was analyzed from the dose–response curves. We also assessed the half-maximal inhibition (IC₅₀) in cardiac cells using propidium iodide dye (IC₅₀ PI) and by measuring disruption in the mitochondrial membrane potential (IC₅₀ MMP). We observed that the most sensitive marker was α-MHC in the case of both Dox and Pac, and the order of sensitivity of the various prediction assays was MMP > protein expression by FC > gene expression by qRT-PCR > cell viability by PI staining. The results could enrich the screening of drug-induced cardiotoxicity in vitro and propose disruption in MMP along with downregulation of α-MHC protein as a potential biomarker of predicting cardiotoxicity earlier during drug safety evaluation.

Keywords

Cardiac biomarker doxorubicin paclitaxel cardiotoxicity in vitro mitochondrial membrane potential α-MHC drug safety

Introduction

As per the Tufts Center for the Study of Drug Development, the cost involved in bringing a drug into the market costs around 2.5 billion US$¹; hence, the focus is to eliminate false lead compounds by using in vitro models of preclinical drug screening. Cardiotoxicity is one of the major reasons behind the withdrawal of the drugs.^2,3 Moreover, with cardiac anomalies being reported in cancer survivors, several years after chemotherapy, there is an urgent need to screen out potentially cardiotoxic drugs and look for cardioprotective strategies which could possibly prevent heart failure.⁴ Late-stage drug-induced toxicity results in substantial financial losses as well as hard work invested during development. Despite recent advances, comprehensive metrics for predicting and monitoring cardiotoxicity are still lacking. One reason could be the choice of testing system as many of these assays use virally transformed animal cells or tumor cell lines. These methods have an inherent flaw as they are not of human origin, nor are they karyotypically normal and structurally are not related to the human heart, thereby generating artefactual data. Alternative methods use genetically and electrophysiologically normal primary cardiomyocytes which are isolated from animal hearts, but as these cells are terminally differentiated, they do not proliferate, their industrial application is difficult, and lab-to-lab variation is seen leading to hazy cardiotoxic responses in humans. Moreover, human heart samples are rarely used, because of limitations in biopsy size/cell numbers, proliferation, uncertain in preparation, and doubtful normal state.^5,6 So, the cell type used for predicting cardiotoxicity in vitro plays an essential role in an early assessment.⁶ To date, embryonic stem (ES) cells have shown their potential in toxicity testing displaying mechanistic understanding of how differentiation of functional cardiac precursor cells from stem cells occurs in vivo.^7,8

In this study, KIND-2 cells, adapted to feeder-free conditions, were differentiated toward cardiac lineage using directed differentiation protocol.⁹ An attempt was made to evaluate the sensitivity of different cardiac endpoints obtained by comparing the relative expression of cardiac lineage biomarkers using quantitative real-time polymerase chain reaction (qRT-PCR) and flow cytometry (FC) with disruption in mitochondrial membrane potential (MMP) in cardiac cells. The endpoints were quantified from dynamic expression of transcription factors that underlie cardiomyocyte differentiation, that is, Nkx2.5, Tbx5, α-myosin heavy chain (α-MHC), and cardiac troponin T (cTnT), which was dependent on detection of mRNA transcripts via qRT-PCR in cardiac precursor cells (CPC). While these cardiac endpoints are highly sensitive and can be obtained using small amounts of mRNA, it does not provide decision at the level of the individual cells. An alternate approach used in this study is to use specific antibodies against Nkx2.5, Tbx5, α-MHC, and cTnT for detection and quantification of population expressing these markers after exposure to drugs using FC.

This study is innovative in the sense that it utilizes the power of specific biomarkers to assess the extent of the damage that can be inflicted on the heart by drugs. In other words, we can anticipate in vitro cardiotoxicity by the development of a cardiac-specific biomarker panel.

Material and method

Maintenance of human ES cell line, KIND-2 under feeder-free conditions

The KIND-2 cell line was a kind gift from Dr Deepa Bhartiya, National Institute for Research in Reproductive Health, Mumbai, India. It was grown under feeder-free conditions on Geltrex (Invitrogen, India)-coated dishes (Invitrogen, India) and maintained in Essential 8 medium (Gibco). Ready to use Geltrex was added to the culture dish in sufficient volume to cover the entire surface area (3 mL for 60 mm dish and 1.5 mL for 35 mm dish) and the plate was incubated at 37°C for at least 2 h. Following this, KIND-2 cells were grown on these dishes. Media was changed after every 24 h until the KIND-2 colonies were large enough and ready for passage. Differentiated colonies were identified (ones which were dark from the center and had excessive fibroblast-like structure at the periphery) and removed by scratching them using the 1 mL tip. Undifferentiated KIND-2 cells maintained in feeder-free conditions were passaged either by manually dissociating them into small clumps and transferred into a new Geltrex-coated dishes or enzymatically Versene solution (Invitrogen) was used during the passage. For manual passaging of feeder-free KIND-2 cells, confluent cells were scraped off the dish using cell lifter (Corning Inc., India) and collected in a sterile 15 mL centrifuge tube. The cell suspension was centrifuged at 1000 r/min for 4–5 min. After removing the supernatant, the cells were resuspended in complete media taking care not to break the clumps too small or leave the clumps too big (this was done by triturating the centrifuged cell suspension with 5 mL disposable steripette serological pipette) and transferred into a new Geltrex-coated dishes. The plates are kept at 37°C in a 5% carbon dioxide incubator. For enzymatic treatment, the cells were washed with phosphate buffer saline (PBS) twice and then treated with Versene solution for few seconds allowing the detachment of colonies. Complete Essential 8 medium was added (twice the amount of Versene solution used). The colonies were collected using 5 mL disposable stripette serological pipette in 15 mL centrifuge tube and centrifuged for 4–5 min at 1000 rpm. The supernatant was discarded, and fresh complete medium was added. Similarly, the trituration was done, and clumps were transferred to new Geltrex-coated dish.

Immunocytochemistry

The cells were fixed with 4% paraformaldehyde for 20 min and then washed with PBS containing 1% bovine serum albumin (BSA) (wash buffer). Then 0.2% triton X was added for 15 min at room temperature. After giving washings with wash buffer, nonspecific binding was blocked with PBS containing 5% BSA for 1 h at room temperature. Then, the cells were incubated with primary antibody (Oct, Sox2, and Nanog from Thermo Scientific Pierce, India) diluted with 1% BSA kept overnight at 4°C. After giving washings with wash buffer, the cells were incubated for 2 h with secondary antibody (Sigma-Aldrich, India) used at the dilution of 1:500 in 1× PBS and 1% BSA. After three washes with wash buffer, the cells were exposed to 1 μg/mL Hoechst dye solution for 20 min. The cells were washed with PBS for 5 min and then viewed under the Nikon Eclipse Ti microscope (Tokyo, Japan) at ×10 magnification.

Assessment of cytotoxicity with test compounds under study

To evaluate changes in metabolism associated with drugs, we treated cardiac cells with a training set of drugs with known toxicity. The cardiotoxic drugs, namely paclitaxel (Pac) and doxorubicin (Dox), were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 0.01 M and stored as single-use aliquots at −20^°C. This stock solution was diluted further in culture media in a 3-fold dilution series. After seeding, the cells were treated for 48 h for cytotoxicity analysis. DMSO (0.2%) served as solvent control. The noncardiotoxic drug penicillin G (Pen G) was dissolved in PBS. This stock solution was diluted further in culture media in a 2-fold dilution series. PBS (0.5%) served as solvent control for Pen G-treated cells.

Directed differentiation of KIND-2 into cardiac lineage

KIND-2 cells, which were maintained under feeder-free conditions in Geltrex-coated dish with Essential 8 medium, were transitioned to serum-free medium RPMI (Gibco) supplemented with 5% B27 supplement (Gibco) and 1% Glutamax (Invitrogen) to initiate directed differentiation. They were treated sequentially with 100 ng/mL Activin A (R&D Systems, USA) and 8 ng/mL basic fibroblast growth factor (Invitrogen) for 24 h followed by 15 ng/mL of human recombinant bone morphogenetic protein 4 for 4 days and then with Wnt inhibitor Dkk1 (Gibco) for additional 4 days. These growth factors have been shown to induce cardiogenic mesoderm from KIND-2 cell line.⁹ Following the treatment, the cells were maintained in the RPMI-B27-Glutamax medium for additional 12 days. On days 4, 8, 15, and 20 of differentiation, cells were collected for RNA isolation.

Quantitative RT-PCR analysis of KIND-2-derived cells

The SYBR green PCR core reagents (Bio-Rad, India) were used for qRT-PCR. Direct detection of PCR reaction products was monitored by measuring the increase in fluorescence caused by binding of SYBR green to double-stranded DNA using Bio-Rad CFX manager software. Amplification was carried out with the primer set recognizing marker genes which were essential for human cardiac differentiation and pluripotency. For directed differentiation expression of genes, Brachyury, Nkx2.5, Mef2C, Tbx5, cTnT, atrial natriuretic factor (ANF), ventricular myosin light chain(MLCv), and α-MHC were assessed at days 4, 8, 15, and 20. The levels of pluripotency genes Oct4, Sox2, and Nanog were also assessed at various time points during differentiation. The Ct values generated by CFX manager software (Bio-Rad) for all samples were normalized with the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase. The 2^−ΔΔCt method was used to calculate the relative fold change in gene expression. The fluorescence emitted at each cycle was captured and melt curve analysis was performed at the end of 40 cycles to determine the homogeneity of amplified products. The primer sequences for qRT-PCR are listed in Supplementary Table S1. For qRT-PCR endpoint analysis, the cardiac cells were seeded on 0.1% gelatin-coated dishes and were treated with different concentrations of Dox (0.72, 2.17, 6.50, and 19.6 µM), Pac (0.09, 0.18, 0.37, and 0.74 µM), and Pen G (250, 500, 1000, and 2000 µg/mL). After 48 h, the RNA was isolated and analyzed.

Intracellular FC for lineage-specific proteins expressed during cardiac differentiation to assess directed differentiation

The percentage of cells positive for expression of cardiac marker proteins expressed during differentiation obtained by directed differentiation of human ES cells at day 20 was analyzed using intracellular FC (BD Accuri C6, USA). The cardiac cells were dissociated into single-cell suspension and then fixed in 4% paraformaldehyde solution in PBS for 20 min. After three washing, the cells were permeabilized with 0.4% triton X-100 solution in PBS for 10 min, followed by incubation with 5% BSA in PBS for 15 min to block the nonspecific binding. This was followed by rinsing with washing buffer for three times and the cells were then incubated with primary antibody (Brachyury, Mesp1, Nkx2.5, Mef2C (Abcam), and Tbx5 (Santa-Cruz, USA) for 1 h on ice. Following this, the cells were incubated with secondary antibody for 15 min and then washed three times using washing buffer, resuspended in 500 µl of PBS along with 500 µl of FACS Flow™ for fluorescence-activated cell sorting analysis. Untreated cells were used as a control. All data analysis was carried out using Cell Quest software (Becton Dickinson, USA).

Assessment of cytotoxicity by PI staining

The cytotoxic effects of test substances on the human ES cells-derived cardiac cells were determined by propidium iodide (PI) dyestaining. KIND-2-derived cardiac cells were plated on gelatin-coated six-well plate and treated with various concentrations Dox (0.24, 0.72, 2.17, 6.5, 19.6, 58.8, and 176 µM), Pac (0.0, 0.08, 0.24 0.74, 2.22, 6.66, and 20 µM), and Pen G (250, 500, 1000, and 2000 µg/mL) for a period of 48 h. The cells were washed with PBS and then trypsinized to obtain single-cell suspension. PI (5 μg/mL) treatment was given for 2–3 min. This was followed by the analysis of live and dead cell population in BD Accuri C6 FC. PI is excited at 488 nm and emits at a maximum wavelength of 617 nm.

Detection of MMP disruption

A distinctive feature of the early stages of apoptosis is the effect on the mitochondria, which includes changes in membrane and redox potential in response to cytotoxic drugs. Human ES cell–derived cardiac cells treated with or without drugs were collected in 15 mL centrifuge tubes and incubated with MitoTracker Red CMXRos according to the manufacturer’s instructions (ThermoFisher Scientific,USA). After a wash in PBS, cells were analyzed by FC for MMP disruption. The cardiac cells at day 20 were seeded on 0.1% gelatin-coated dishes and treated for 48 h with different concentrations of Dox (0.24, 0.72, 2.17, 6.5, 19.6, 58.8, and 176 µM), Pac (0.02, 0.08, 0.24, 0.74, 2.22, 6.66, and 20 µM), and Pen G (31.25, 62.5, 125, 250, 500, 1000, and 2000 µg/mL).

Intracellular FC for cardiac biomarkers expressed during cardiac differentiation to assess cardiotoxicity after exposure to drugs under study

The cardiac cells were seeded on 0.1% gelatin-coated dishes and were treated with different concentrations of Dox (0.72, 2.17, 6.50, and 19.6 µM), Pac (0.09, 0.18, 0.37, and 0.74 µM), and Pen G (250, 500, 1000, and 2000 µg/mL). After 48 h, the expression of cardiac biomarker was analyzed by intracellular FC as described above.

Statistical analysis

All data are presented as the mean ± standard deviation from three independent experiments done in triplicates. The IC₅₀ values were calculated using dose–response curves. Statistical analysis was assessed by one-way analysis of variance followed by a Dunnett’s multiple comparisons test using GraphPad Prism, version 7. For all graphs, an asterisk indicates a significant difference with the untreated group (*p < 0.05).

Results

Pluripotency characterization

To have an effective cardiac differentiation, the starting material, that is, the stem cells have to be pluripotent. We verified the pluripotent potential of the KIND-2 cells grown and maintained under feeder-free conditions by alkaline phosphatase staining and immunocytochemistry for pluripotent markers, namely Oct4, Sox2, and Nanog. The expression analysis revealed KIND-2 cells retained the expression of alkaline phosphatase and pluripotency markers as shown in Figure 1.

Figure 1.

(a) Characterization of pluripotent expression in KIND-2 cells by alkaline phosphatase staining (magnification: ×4, bars: 100 µM) and its (b) phase contrast pictograph (magnification: ×4, bars: 100 µM). Immunostaining of human ES colony showing the expression of pluripotent markers, Oct4 (f), Sox2 (g), and Nanog (h) (magnification: ×10, bars: 100 µM). The corresponding phase contrast images of ICC are (c), (d), and (e). (i) Analysis of molecular determinants expressed during cardiogenesis using qRT-PCR. The expression level of Brachyury, Mef2c, Mlc-2a, ANF, α-MHC, Nkx2.5, and cTnT was normalized against GAPDH. The y-axis represents relative fold change of expression. Bar graphs show means ± SD (n = 3). Significant changes in cardiac cells (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001). (j) FC analysis depicting percentage of cells expressing Brachyury (i, ii, iii), Mesp1 (iv, v, vi), Tbx5 (vii, viii, ix), Mef2C (x, xi, xii), and Nkx2.5 (xii, xiii, xiv) within the cardiac precursor population at various days during directed differentiation of KIND-2 cells. ES: embryonic stem; SD: standard deviation; qRT-PCR: quantitative real-time polymerase chain reaction; ANF: atrial natriuretic factor; α-MHC: α-myosin heavy chain; cTnT: cardiac troponin T; GAPDH: glyceraldehyde 3-phosphate dehydrogenase.

Molecular determinants expressed in cardiogenesis analyzed by qRT-PCR

In vitro-directed cardiac differentiation using a cocktail of growth factors revealed efficient cardiac differentiation. It was noted that the KIND-2 cells proceeded toward committed mesoendoderm lineage as Brachyury expression was more than 140-fold at day 4. This expression decreased to less than 10-fold on day 8 and on day 20, the expression was almost undetectable.

The expression of early cardiogenic transcript Mef2C was upregulated on day 8 and remained elevated till day 15. The expression of cardiac sarcomere protein Mlc-2a reached its peak on day 20, that is, 37-fold. Similarly, ANF showed peak expression at day 20, that is, 25-fold. The cardiac transcription factor Nkx2.5 was significantly elevated (41-fold) at day 4. Nkx2.5 transcripts increased to 60-fold at day 8 and 65-fold expression at day 15 and reached its maximum at day 20, that is, 77-fold. There was a continuous increase in the expression of myofilament protein α-MHC from day 8 of differentiation and showed a maximum on day 20, that is, 63-fold. cTnT was readily detectable with peaked expression of 206-fold at day 8 of differentiation. At day 20, the expression was found to be 82-fold (Figure 1(i)). The presence of cardiomyocytes was seen easily by microscopic observation of spontaneously contracting regions.

Quantitating in vitro cardiogenesis via protein expression

Cardiogenesis was studied using directed differentiation method for deriving cardiac population. The observed data of protein pattern during cardiogenesis during directed differentiation were normal. The first event related to cardiogenesis was a peak in the expression of primitive streak, mesoderm, and cardiomesoderm markers (Brachyury and Mesp1) during differentiation. As shown in Figure 1, the percentage of Brachyury was 52.2% at day 4 (Figure 1(j)-(i)) and that of Mesp1 was 52.1% at day 12 (Figure 1(j)-(v)). The expression of first cardiac field marker Tbx5 showed maximum population at day 20, that is, 83% (Figure 1(j)-(ix)). Apart from this established transcription factor, the population of cardiac precursor from KIND-2 also expressed another cardiac transcription factor, that is, Mef2C and it was seen that at day 4, the 74.6% of the cell population expressed this (Figure 1(j)) and day 12 (Figure 1(j)) 69.4% expression was noted. Further cardiac differentiation was tracked by expression of cardiac-associated transcription factors Nkx2.5, which showed highest expression at days 4 (Figure 1(j)-(xiii)) and 12 (Figure 1(j)-(xiv)), that is, 75.1% and 74.2%, respectively.

Cardiotoxicity analysis using PI staining

Maintenance of cardiac cell morphology is fundamental to its basal function. Cardiotoxicity due to intake of drugs can be the result of severe alteration in the mechanical function of the heart (functional toxicity), or morphological changes which would include loss of cellular/subcellular components of the heart or loss of membrane integrity (structural toxicity). Any drug or intervention which could cause damage to membrane of cardiac precursor cells would also affect the selective permeability of the membrane, thereby allowing the entry of PI. Therefore, cardiomyocytes treated with Pac, Dox, or Pen G were analyzed by FC using PI which is a cell impermeant dye which is excluded by viable cells having an intact membrane. The analysis revealed that cardiotoxic drugs, Pac and Dox, showed dose-dependent responses in KIND-2-derived cardiac cells (Figure 2(a)). FC analysis showed 58% live cell population in cardiac cells treated with 6.5 µM Dox, 50.2% live cell population in cells treated with 19.6 µM Dox, and the live cell population dropped to 26.7% upon 176 µM Dox treatment. Pac treatment of cardiac cells showed on exposure to 0.74 µM, 60.2% live population, which dropped to 27.6% upon treatment with 20 µM Pac. In the cells treated with Pen G, the cell death was observed at only higher concentrations (Figure 2). Based on these results, the IC₅₀ PI for Dox which was seen to be 19 µM, 1.78 µM in the case of Pac, and 820 µg/mL for Pen G (Figure 2(b)).

Figure 2.

Human ES cell–derived cardiac cells at day 20 of directed differentiation were placed on gelatin-coated dishes treated with cardiotoxic drugs, Pac and Dox, and the noncardiotoxic drug, Pen G, for 48 h. (a) Cell viability analyzed using PI staining using BD Accuri C6 FC. (b) Dose–response curves of the cardiac cells upon treatment. Significant changes between drug-exposed compared to vehicle control, cardiac cells (*p < 0.05, **p < 0.01, ***p < 0.001). ES: embryonic stem; Dox: doxorubicin; Pac: paclitaxel; Pen G: penicillin G; PI: propidium iodide.

Analysis of MMP disruption (endpoint)

Oxidative stress and cellular dysfunction at the level of the mitochondria is a common mechanism caused by cardiotoxic drugs. This makes the MMP ideal for predicting structural toxicity. Based on its role in cardiotoxicity caused by drugs, MMP can be used as an endpoint for predicting the cardiotoxicity. We analyzed the disruption in MMP in KIND-2-derived cardiac population following exposure to Pac, Dox, and Pen G. The cells showed disruption of MMP with increasing concentrations of Pac and Dox. The IC₅₀ MMP obtained for Dox was 0.32 µM and for Pac was 0.07 µM (Table 1). For noncardiotoxic drug Pen G, the disruption was observed only at higher concentration of Pen G beyond 500 µg/mL (Table 1).

Table 1.

Alterations in the MMP of KIND-2-derived cardiac cells after exposure to the drugs.

Dox (µM)		Pac (µM)		Pen G (µg/mL)
Concentration	Disruption of MMP	Concentration	Disruption of MMP	Concentration	Disruption of MMP
0.24	48.3 ± 5%	0.02	40.1 ± 6.3%	31.25	6.6 ± 2.7%
0.72	68.3 ± 7.3%	0.08	70.3 ± 4.2%	62.5	7.0 ± 2.1%
2.17	72.5 ± 6.4%	0.24	76.0 ± 2.7%	125	11.4 ± 1.4%
6.5	90.6 ± 2.0%	0.74	87.3 ± 1.6%	250	23.6 ± 3.6%
19.6	92.6 ± 1.6%	2.22	88.8 ± 3.6%	500	41.5 ± 5.2%
58.8	93.6 ± 2.4%	6.66	90 ± 2.0%	1000	83.1 ± 2.0%
176	94.1 ± 1.3%	20	98.7 ± 0.6%	2000	85.6 ± 3.5%

MMP: mitochondrial membrane potential; Dox: doxorubicin; Pac: paclitaxel; Pen G: penicillin G.

New endpoints evaluated for Dox, Pac, and Pen G using cardiac biomarker

The expression of various cardiac factors, namely Nkx2.5, Tbx5, α-MHC, and cTnT, was analyzed for obtaining novel endpoint for prediction of cardiotoxicity for Dox and Pac (Figure 3,4). The drug induced dose-dependent deviation in their expression at both the mRNA and the protein level. The half-maximal inhibition (IC₅₀) in the expression both at gene and at protein level yielded useful data which could be considered as critical endpoints to determine cardiotoxicity (Table 2). The relative fold change in gene expression was well correlated with increasing drug exposure, that is qRT-PCR IC₅₀. The decrease in population of cells expressing cardiac proteins was plotted against the concentration of the drug (FC IC₅₀). The analysis of the results obtained using the various endpoints point toward the greater sensitivity of protein expression when the cells were exposed to the drugs.

Figure 3.

qRT-PCR and FC analysis of cardiac marker Nkx2.5, Tbx5, and α-MHC following drug treatment. The expression pattern of all the cardiac markers was disturbed by cardiotoxic drugs, Dox and Pac. The y-axis represents fold change of expression of the studied gene compared with the vehicle control, bar graphs show means ± SD (n = 3). For the flow cytometry analysis,the y-axis represents % population of cells expressing the specific protein. Significant changes between drug-exposed and vehicle control, cardiac cells (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001). qRT-PCR: quantitative real-time polymerase chain reaction; FC: flow cytometry; α-MHC: α-myosin heavy chain; Dox: doxorubicin; Pac: paclitaxel; SD: standard deviation.

Figure 4.

qRT-PCR and FC analysis of cardiac marker Tbx5 and cTnT following drug treatment. The expression pattern of all the cardiac markers was disturbed by cardiotoxic drugs, Dox and Pac. The y-axis represents fold change of expression of the studied gene compared with the vehicle control, bar graphs show means ± SD (n = 3). For the flow cytometry analysis,the y-axis represents % population of cells expressing the specific protein. Significant changes between drug-exposed and vehicle control, cardiac cells (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001). qRT-PCR: quantitative real-time polymerase chain reaction; FC: flow cytometry; cTnT: cardiac troponin T; Dox: doxorubicin; Pac: paclitaxel; SD: standard deviation.

Table 2.

IC₅₀ values for the 10 endpoints of the cardiotoxicity determined from dose–response curves for all three compounds tested.

Drug	IC₅₀ PI	IC₅₀ MMP	IC₅₀ Nkx2.5 qRT-PCR	IC₅₀ Nkx2.5 (FC)	IC₅₀ Tbx5 qRT-PCR	IC₅₀ Tbx5 (FC)	IC₅₀ α-MHC qRT-PCR	IC₅₀ α-MHC (FC)	IC₅₀ cTnT qRT-PCR	IC₅₀ cTnT (FC)
Dox (µM)	19.0	0.32	5.1	2.11	15.6	3.9	4.2	1.4	17.69	2.37
Dox (µM)	1.78	0.07	0.62	0.19	0.64	0.28	0.2	0.08	0.67	0.12
Pen G (µg/mL)	820	814.1	^a	1583	^a	^a	1550	1604	857.5	1477

MMP: mitochondrial membrane potential; Dox: doxorubicin; Pac: paclitaxel; Pen G: penicillin G; PI: propidium iodide; qRT-PCR: quantitative real-time polymerase chain reaction; FC: flow cytometry; α-MHC: α-myosin heavy chain; cTnT: cardiac troponin T.

^aIC₅₀ could not be assessed as even with the highest exposure (concentration) of Pen G, the expression of various endpoints did not go beyond 50%.

Discussion

Heart tissue homeostasis at the physiological as well as pathological level is maintained by the cardiac precursor cells which give rise to the various cell types such as cardiomyocytes, smooth muscle cells, and endothelial cells. Any insult such as drugs to these cardiac cells would lead to senescence and could contribute to either acute or late manifestation of cardiotoxicity. It has been suggested that treatment with certain drugs such as anthracyclines could leave a cellular signature in the heart which could manifest itself at a later point resulting in deleterious effects.⁴

Therefore, in this present study, we used a training set of two cardiotoxic drugs, that is, doxorubicin and paclitaxel, and assessed their effects on the cardiac cells through various assays, so as to come up with certain indicators which could be evaluated for potential cardiotoxic effects. One of the earliest and most-effective anthracycline drugs is doxorubicin, isolated from the bacterium Streptomyces peucetius. Owing to its efficacy, its use has increased in terms of conditions which can be treated, and it is also being administered in higher dosages to improve the clinical outcome. Paclitaxel is a plant-derived drug which was first isolated from the bark of the Pacific yew tree. It is used for the treatment of various cancers and acts by binding to the tubulin cytoskeletal protein, thereby preventing cell transition in the G2/M phase and inducing apoptosis. Although both these drugs are indispensable to cancer treatment, they, however, contribute to the ensuing cardiomyopathy which has become an established side effect of their use in many patients. The most common cardiac adverse effect associated with these drugs are arrhythmia and cardiac ischemia.^10,11

Drug-related cardiac toxicity may be caused via several cellular mechanisms, such as the production of reactive oxygen species (ROS) and their subsequent binding, leading to damage to DNA, inhibition of topoisomerases, perturbation of calcium homeostasis, and mitochondrial damage and eventually cell death through apoptosis or necrosis.^12

–16 Dox-induced cardiotoxicity is highly dose-dependent and based on onset of toxicity has been categorized as acute, early, or late.¹² To assess the toxicity induced by chemical insults, the loss of cellular viability has been reported to be the most sensitive endpoint following exposure to drugs.¹⁷ The generation of free radicals, leads to peroxidative damage to the cardiac cell membranes and results in influx of intracellular calcium and ensuing loss in viability. Mitochondrial dysfunction also has been reported, which correlates with morphologic changes seen in cardiotoxicity, due to chemotherapeutic drugs.¹⁸ However, there are various steps prior to the irreversible loss of viability which if detected earlier and at lower drug exposure, could possibly prevent widespread tissue damage resulting in serious and sometimes life-threatening morbidities. Moreover, screening drugs for potential cardiotoxicity using a panel of markers would provide a robust tool to anticipate and possibly prevent serious cardiac damage in the future, as manifestation of cardiotoxicity several years after treatment has been noted in cancer survivors .Therefore, in our present study, we utilized human ES cell-derived cardiac cells as a screening tool and assessed the effects of certain cardiotoxic drugs on the MMP along with alterations in the expression of cardiac markers both at the gene and at the protein level, with an aim to obtain a selective cardiotoxic endpoint that could be proposed as an in vitro index to predict cardiotoxic effects of drugs. The results indicated differential sensitivity to Dox and Pac exposure even at lower concentrations by various approaches.

In drug safety studies, established toxicity biomarkers are used along with other standard data to determine dose-limiting organ toxicity and to define species sensitivity for new chemical entities intended for possible use as human medicines. Ideally, a biomarker should be reproducible and accurately predict the incidence of outcome or disease. The use of cardiac biomarker(s) has been incrementally increasing in the in vitro assay setting; though, additional data are required to implement these biomarkers for detection, management, and ultimately averting cardiotoxicity at an early stage, before the damage becomes irreparable. In our study, selection of these markers was based on their role in cardiac functioning. The stipulation of specific molecular, cellular, and physiological inputs by developing cardiomyocytes yields essential building blocks necessary to put together a complete functional cardiac system. In the light of this, we selected key players which impact cardiac differentiation and function and studied alterations in response to drugs, using various assays which could detect the changes.

The cardiac transcription factor, Nkx2.5, is one of the earliest transcription factors expressed during cardiogenesis and is known to be abundantly expressed in the adult heart. Nkx2.5 is a prerequisite for terminal differentiation and morphogenesis of the early developing heart.¹⁹ Tbx5 is another transcription factor which has also been shown to be expressed in the developing heart. It plays an important role in developing the cardiac septum as well as the electrical system which bring together the contractions of the cardiac muscles and its presence would signify a cardiac cell precursor population.²⁰ The functional myocardium comprises three major types of proteins which are myofibrillar, metabolic, and extracellular.²¹ Several myofibrillar proteins that are specifically expressed by the cardiac cells can serve as markers of cardiomyocytes, one of which is cTnT.²² The expression of α-MHC regulates a cardiac muscle specifically involved in active force generation and maintains the cardiac cell lineage.^23,24 These proteins can serve as biometric measurements which could possibly have the potential for critical quantitative information about the biological condition of heart system after drug exposure.^8,9 Therefore, it is essential to study the changes at the structural as well as genetic level which could be used as a blueprint to suggest possible cardiotoxicity and take remedial measures.

The viability cells of the cardiac lineage, derived from directed differentiation of human ES cells following treatment with Dox and Pac, were studied by staining the cells with the vital dye PI. Since the available data for Dox and Pac confirm their cell toxic potential, assessment of viable cells after exposure served as a useful in vitro endpoint. However, at lowered concentration of these drugs, PI analysis did not reflect significant changes in viability as compared to controls, thereby suggesting lack of sensitivity.

Mitochondrial dysfunction, which encompasses various pathways, has been shown to be a key player in Dox- and Pac-mediated cardiotoxicity.^12,15,25,26 Owing to the large number present in the cardiac cells, mitochondria can be considered as major players in the development of cardiotoxicity. This coupled with the fact that mitochondria account for the majority of adenosine triphosphate (ATP) production in cardiomyocytes by mitochondrial oxidative phosphorylation, disruption of mitochondrial function therefore, would impact the overall viability of these cells. Therefore, we assessed the disruption of MMP as an early indicator for cardiotoxicity using the training set of Dox and Pac and experimentally validated whether MMP could serve as a sensitive endpoint. Dose-dependent analysis revealed drug concentration exposure was effectively correlated with MMP. Reports have shown that paclitaxel could act directly on isolated mitochondria from human neuroblastoma cells to induce the permeability transition pore-dependent release of mitochondrial cyt c.^27,28 In the light of these observations, Pac could be acting in the cardiac cells to disrupt the MMP resulting in the observed toxicity to the mitochondria. The results of our study point to the effect of Pac on the mitochondria by causing a disruption of the MMP, probably due to the effect of Pac on mitochondrial tubulin which is associated with the opening of the voltage-dependent anion channel, thereby leading to cardiotoxicity. We, however, cannot comment whether mitotic catastrophe was also observed as we did not carry out experiments to evaluate it. Moreover, as we used relatively lower concentrations of Dox and Pac (in the micromolar range), the effects observed could be more reliably correlated with the in vivo setting. However, the exact mechanism(s) responsible for Dox- and Pac-induced cardiotoxicity remains unclear and several hypotheses have been proposed in past decade.²⁹ It is reported that the alpha isoform of MHC not only has greater requirement for ATP but, also has elevated ATPase activity and is therefore the major contributor to heart contraction. The presence of injured mitochondria and their disorganization have been also detected in cardiac tissue of humans exposed to doxorubicin.³⁰ Since mitochondrial dysfunction may also affect the gene and protein panorama,³¹ we studied whether the ensuing cardiac toxicity would impact expression of key genes and proteins which may result in irreparable damage to the cardiac cells.^25,32 In a recent study, Zhang et al. emphasized the role of the mitochondria in Dox exposure and they showed that the Dox-induced DNA double-strand breaks and transcriptome alterations in cardiomyocytes resulted in flawed function of mitochondria and ROS production, however deletion of Top2b diminished the damage.³¹

In established studies for drug safety, biomarkers can be used as baseline data to predict dose–responses for toxicity and to define species sensitivity for new chemical entities intended for possible use during the treatment. The mechanism responsible for restricting expression of these cardiac biomarkers after exposure to the drugs could provide clues for the identification and regulatory events involved in the functioning and maintenance of the cardiac system. It has been reported that Dox downregulates expression of a number of cardiac muscle-specific contractile proteins, which includes MHC, cTnT, and several mitochondrial proteins. However, studies conducted using cardiomyocytes derived from ES cells revealed that in the case of late onset doxorubicin-induced cardiomyopathy, cTnT release might not be the most optimal biomarker.³³ In various studies following treatment with anthracyclines, cTnT did not predict subsequent development of cardiac dysfunction.^34,35 Global transcriptional profiles obtained through microarray analysis revealed clusters of genes that were differentially expressed during doxorubicin exposure in cardiomyocytes. Reports have suggested that lowered cardiac muscle proteins have a direct bearing on the contractile ability of cardiomyocytes which could be a plausible reason for the pathologic findings seen in DOX-induced cardiomyopathy.³⁶

The panel of endpoints reported in this work using molecular and protein expression approaches for Dox- and Pac-induced cardiotoxicity may provide insights of possible cardiotoxic behavior of other drugs. In a recent study, induction of cardiotoxicity by Carfilzomib was linked to significantly downregulated α-MHC mRNA expression.³⁷ Mitochondria have an important role in energy production in cardiac cells, apart from other cellular process like homeostasis, free radical production, and ultimately cell death. Specifically, in the adult cardiac cell, mitochondria produce 90% of ATP and occupy 30% of cardiac cell mass. This interlinking establishes the challenging function of the cardiac system which constantly need a fast and efficient supply for intracellular energy delivery to the ATP consumers for rhythmic contractions of the heart throughout life.^38,39 As reported in our study, the decrease in the expression of the cardiac marker α-MHC at both the mRNA and the protein level could potentially serve as an useful endpoint for more rigorous prediction during the process of drug development. Moreover, by the incorporation of additional techniques such as quantitative, qRT-PCR, and FC expression analyses, our results are a modest effort to put forth experimental evidence for quantifiable and sensitive endpoints for predicting cardiotoxicity. Importantly, the analysis of the additional endpoints using a larger panel of gene and protein expression may uncover false-negative results of in vitro observation.

In conclusion, the observations in this exploratory study put forward experimental proof of additional cardiac biomarker-based endpoints to bridge analytical data and could be used to generate predictive cardiac toxicity data, based on multiple parameters. However, to validate the usefulness of these endpoints, additional studies need to be carried out with a larger subset of drugs for the establishment of an integrated model for tracking hitherto unknown cardiotoxic effects at affordable cost so that maximal benefit with lowered long-time cardiotoxic side effects can be passed on to the consumer.

Supplemental material

supplementary_table_primers - Disruption of mitochondrial membrane potential coupled with alterations in cardiac biomarker expression as early cardiotoxic signatures in human ES cell–derived cardiac cells

supplementary_table_primers for Disruption of mitochondrial membrane potential coupled with alterations in cardiac biomarker expression as early cardiotoxic signatures in human ES cell–derived cardiac cells by Saras Jyoti and Simran Tandon in Human & Experimental Toxicology

Footnotes

Acknowledgement

The authors are grateful to Dr Deepa Bhartiya, National Institute for Research in Reproductive Health, Mumbai, India, for providing the KIND-2 cells.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was financially supported by the Department of Biotechnology (DBT), Project No. 102/IFD/SAN/428/2013-2014, Ministry of Science and Technology, Government of India.

Supplemental material

Supplemental material for this article is available online.

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